Journal: Genes & Diseases

Article Title: TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

doi: 10.1016/j.gendis.2024.101251

Figure Lengend Snippet: Inhibition of FSP1 fails to induce ferroptosis. (A – C) Rat NP cells were treated with or without TNFα and RNA sequencing was performed. The differentially expressed genes were presented in the volcano plot. Inflammatory response and TNF signaling pathway were activated on GSEA. (D) FSP1 was up-regulated after TNFα stimulation. (E, F) Transcriptional levels of FSP1 and GPX4 in normal NP cells. (G) Cell viability of NP cells with different concentrations of iFSP1 treatment. (H) Cell morphology after treatment with different doses of iFSP1 (0, 1, 2, 5, 10, and 20 μM). Scale bar, 200 μm. (I, J) Cell apoptosis of NP cells after treatment with different concentrations of iFSP1 (0, 1, 2, 5, 10, and 20 μM) and quantification of apoptotic cells was performed by flow cytometry. (K, L) Live-death staining in NP cells with stimulation by different doses of iFSP1 and death rate (%) calculation. Scale bar, 200 μm. (M) Cell morphology of transmission electron microscope in control and iFSP1 treated groups. Low, 5 μm; high, 1 μm. (N, O) Western blot of FSP1 and GPX4 expression in rat NP cells with stimulation by different doses of iFSP1 and their quantification. (P) Schematic diagram of the role of FSP1 and GPX4 in fighting against ferroptosis in NP cells. FSP1, ferroptosis suppressor protein 1; NP, nucleus pulposus; TNFα, tumor necrosis factor alpha; GPX4, glutathione peroxidase 4. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The apoptotic rate of each group was performed by an Annexin V-FITC/PI Apoptosis Detection Kit (A211, Vazyme, Nanjing, China).

Techniques: Inhibition, RNA Sequencing Assay, Flow Cytometry, Staining, Transmission Assay, Microscopy, Control, Western Blot, Expressing

Journal: Genes & Diseases

Article Title: TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

doi: 10.1016/j.gendis.2024.101251

Figure Lengend Snippet: FSP1-evoked apoptosis by TNFα is related to caspase 3 activation and mitochondrial damage. (A, B) TNFα stimulated rat NP cells were co-cultured with or without iFSP1 and RNA sequencing was performed. The differentially expressed genes were presented in the volcano plot and the TNF signaling pathway was inhibited on GSEA after iFSP1 treatment. (C) The heat map revealed no significance of ferroptotic genes between the two groups, while apoptotic genes containing caspase 3 and caspase 4 were both down-regulated. (D, E) Western blot analysis of FSP1, OPA1, Drp1, Mfn1, Mfn2, Bax, Bcl2, pro-caspase 3, and cleaved caspase 3 in TNFα stimulated rat NP cells with or without iFSP1 treatment and their quantification. (F) Transmission electron microscope of TNFα stimulated rat NP cells with or without iFSP1 treatment. Low, 5 μm; high, 1 μm. (G) JC-1 staining of NP cells of indicated three groups. Scale bar, 20 μm. (H) Caspase-3 GreenNuc™ staining and caspase-3 immunofluorescence in TNFα stimulated rat NP cells with or without iFSP1 treatment. Scale bar, 100 μm. (I) Annexin V/PI staining of three groups and apoptotic rate (%) quantification. FSP1, ferroptosis suppressor protein 1; NP, nucleus pulposus; PI, propidium iodide; TNFα, tumor necrosis factor alpha; ns, not significance. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The apoptotic rate of each group was performed by an Annexin V-FITC/PI Apoptosis Detection Kit (A211, Vazyme, Nanjing, China).

Techniques: Activation Assay, Cell Culture, RNA Sequencing Assay, Western Blot, Transmission Assay, Microscopy, Staining, Immunofluorescence

Journal: Genes & Diseases

Article Title: TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

doi: 10.1016/j.gendis.2024.101251

Figure Lengend Snippet: Fsp1 knockdown reduces cellular reactivity to TNFα and dampens the apoptotic phenotype. (A) Cell morphology in normal NP cells or after siFSP1 transfection. (B, C) Western blot analysis of FSP1 and GPX4 after three times of siFSP1 transfection, and their quantification. (D, E) Western blot analysis of FSP1 in TNFα stimulated NP cells with or without siFSP1 transfection, and its quantification. (F, G) Western blot analysis of Bax, Bcl2, pro-caspase 3, and cleaved caspase 3 in TNFα stimulated NP cells with or without siFSP1 transfection, and their quantification. (H) Caspase-3 GreenNuc™ staining in TNFα stimulated rat NP cells with or without siFSP1 transfection. Scale bar, 100 μm. (I) Annexin V/PI staining in TNFα stimulated rat NP cells with or without siFSP1 transfection, and apoptotic rate (%) quantification. (J, K) Western blot analysis of OPA1, Drp1, Mfn1, and Mfn2 in TNFα stimulated NP cells with or without siFSP1 transfection, and their quantification. (L) Transmission electron microscope of TNFα stimulated rat NP cells with or without siFSP1 transfection. Low, 5 μm; high, 1 μm. (M) JC-1 staining of NP cells of indicated two groups. Scale bar, 20 μm. FSP1, ferroptosis suppressor protein 1; PI, propidium iodide; TNFα, tumor necrosis factor alpha; GPX4, glutathione peroxidase 4; ns, not significance. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The apoptotic rate of each group was performed by an Annexin V-FITC/PI Apoptosis Detection Kit (A211, Vazyme, Nanjing, China).

Techniques: Knockdown, Transfection, Western Blot, Staining, Transmission Assay, Microscopy

Journal: Genes & Diseases

Article Title: TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

doi: 10.1016/j.gendis.2024.101251

Figure Lengend Snippet: Exogenous FSP1 is incapable of triggering cell death and unable to potentiate TNFα-derived cell demise. (A) Cell morphology of NP cells after treatment of different recombinant FSP1 concentrations (0, 10, 20, 50, 100, and 200 ng/mL). Scale bar, 200 μm. (B) Cell viability of NP cells after treatment of different recombinant FSP1 concentrations. (C, D) Annexin V/PI staining in rat NP cells with addition of different doses of FSP1, and apoptotic rate (%) quantification. (E, F) Live-death staining in NP cells with stimulation by different doses of FSP1, as well as death rate (%) calculation. Scale bar, 100 μm. (G) Cell morphology of TNFα (20 ng/mL) stimulated NP cells after treatment of different recombinant FSP1 concentrations (0, 20, 50, 100, and 200 ng/mL). Scale bar, 200 μm. (H) Cell viability of TNFα stimulated NP cells after treatment of different recombinant FSP1 concentrations. (I, J) Annexin V/PI staining in TNFα stimulated rat NP cells with addition of different doses of FSP1, and apoptotic rate (%) quantification. (K, L) Live-death staining of TNFα stimulated NP cells with stimulation by different doses of FSP1 and death rate (%) calculation. Scale bar, 100 μm. FSP1, ferroptosis suppressor protein 1; PI, propidium iodide; TNFα, tumor necrosis factor alpha; ns, not significance.

Article Snippet: The apoptotic rate of each group was performed by an Annexin V-FITC/PI Apoptosis Detection Kit (A211, Vazyme, Nanjing, China).

Techniques: Derivative Assay, Recombinant, Staining

Journal: Frontiers in Oncology

Article Title: A novel compound, SYHA1813, inhibits malignant meningioma growth directly by boosting p53 pathway activation and impairing DNA repair

doi: 10.3389/fonc.2025.1522249

Figure Lengend Snippet: SYHA1813 arrested the cell cycle and induced cell apoptosis and senescence in meningioma cells. (A–D) SYAH1813 arrested cell cycle in IOMM-Lee and CH157 cells. (A, C) Representative flow cytometry analysis and quantitative analysis results of the cell cycle in IOMM-Lee and CH157 cells treated with 2.5, 5, and 10μM SYHA1813 or 0.1% DMSO for 48h before stained with PI; (B, D) Western blotting showed the protein expression of cell cycle-related proteins, Cyclin B1, Cyclin A2, and Cyclin E1, in IOMM-Lee and CH157 cells under the same treatment conditions; (E–H) SYAH1813 induced cell apoptosis in IOMM-Lee and CH157 cells. (E, G) Representative flow cytometry analysis and quantitative analysis results of the cell apoptosis in IOMM-Lee and CH157 cells treated with 2.5, 5, and 10μM SYHA1813 or 0.1% DMSO for 48h before stained with Annexin V-FITC/PI. (F, H) Western blotting showing the protein expression of apoptosis-related proteins, Procaspase3, Caspase3, Bax, and Bcl2, in IOMM-Lee and CH157 cells under the same treatment conditions. (I, J) β-galactosidase staining of IOMM-Lee and CH157 cells treated with 2.5, 5, and 10μM SYHA1813 or 0.1% DMSO for 48h. Senescent cells were identified as blue-stained cells. Representative images of senescent phenotype of cells (left panel). Quantification of the proportion of senescent cells (Blue + cells/Total cells) in IOMM-Lee and CH157 cells (right panel). Scar bar =200 μm. (I) IOMM-Lee; (J) CH157. All data shown represent the mean ± SD. Compared with the control, ***p < 0.001.

Article Snippet: The next day, cells were treated with different concentrations of SYHA1813 (2.5, 5, and 10 μM) or 0.1% DMSO for 48 h. The apoptosis assay was performed according to the Annexin V-FITC/PI Apoptosis Kit’s (Multi Science, China) manufacturer’s instructions.

Techniques: Flow Cytometry, Staining, Western Blot, Expressing, Control

Journal: Frontiers in Oncology

Article Title: A novel compound, SYHA1813, inhibits malignant meningioma growth directly by boosting p53 pathway activation and impairing DNA repair

doi: 10.3389/fonc.2025.1522249

Figure Lengend Snippet: The therapeutic effect of SYAH1813 on meningioma in vivo . Xenograft mouse models were established with IOMM-Lee cells. At 3 days post-implantation, mice were treated with SYHA1813 at the dose of 10 mg/kg body weight or vehicle orally every day. (A) Tumor volume curves of mice in the vehicle and SYHA1813 treatment groups. (B, C) The tumors of xenograft mice were measured on the 21st day. (B) The resected tumor morphology image of mice treated with SYHA1813 or vehicle; (C) the weights of tumors. (D) Hematoxylin and eosin (H&E) staining in tumor sections of mice treated with SYHA1813 or vehicle. (E) Immunohistochemical staining for Ki-67 in tumor sections of mice treated with SYHA1813 or vehicle. Representative images of Ki-67-stained tumor sections (right panel). Quantification of the percentage of Ki67 positive cells (left panel). (F, G) Immunohistochemical staining for γH2AX, and P53 in tumor sections of mice treated with SYHA1813 or vehicle. (H) Schematic diagram of SYHA1813 exerting antitumorigenic effects in meningioma cells. SYHA1813 suppressed meningioma cells proliferation and induced cell cycle arrest, cell apoptosis and senescence, and inhibited meningioma cells growth in vivo . Mechanistically, SYHA1813 activated p53 via ATM/CHK signal pathway in meningioma cells. SYHA1813 further hampered DNA damage repair in meningioma cells. All data shown represent the mean ± SD. Compared with the control, **p < 0.01, ***p < 0.001.

Article Snippet: The next day, cells were treated with different concentrations of SYHA1813 (2.5, 5, and 10 μM) or 0.1% DMSO for 48 h. The apoptosis assay was performed according to the Annexin V-FITC/PI Apoptosis Kit’s (Multi Science, China) manufacturer’s instructions.

Techniques: In Vivo, Staining, Immunohistochemical staining, Control

Journal: Cell Death & Disease

Article Title: 4EBP1 senses extracellular glucose deprivation and initiates cell death signaling in lung cancer

doi: 10.1038/s41419-022-05466-5

Figure Lengend Snippet: A , B A549 and H1299 cells transfected with either control (CTL) siRNA or 4EBP1 siRNAs ( A ), A549 and H1299 cells were transfected with CTL siRNAs or 4EBP1 siRNA or cotransfected with CTL siRNAs and mutant plasmid eIF4E-S209E ( B ). Cells were treated with normal or glucose starvation. Cell growth rate was assessed by MTT assay. The data represent the averages of three independent experiments (mean ± SD). * P < 0.05, ** P < 0.01, *** P < 0.001. C A549 cells transfected with either control siRNA or 4EBP1 siRNAs were treated with normal or glucose starvation for 16 h. The changes of cell morphological was investigated (scale bar = 100 µm, magnification: ×100). D A549 cells transfected with either control siRNA or 4EBP1 siRNAs were treated with normal or glucose starvation for 16 h. Cell apoptosis analysis was done by flow cytometry. E A549 cells transfected with either control siRNA or 4EBP1 siRNAs were treated with normal (0 h) or glucose starvation. The expression levels of the apoptosis related proteins were checked by western blot. C-Caspase3: Cleaved-Caspase3. F , G parental A549 (CON) and A549 stable cell lines with 4EBP1 knockdown (A549-sh4EBP1) were treated with normal or glucose starvation for 12 h. Lactic acid levels were monitored ( F , upper panel). ECAR ( F , lower panel) and OCR ( G ) measurements were done.

Article Snippet: Flow cytometric analysis of apoptotic cells was performed using the Annexin V-FITC / PI Apoptosis Detection Kit (Elabscience).

Techniques: Transfection, Control, Mutagenesis, Plasmid Preparation, MTT Assay, Flow Cytometry, Expressing, Western Blot, Stable Transfection, Knockdown

Journal: Cell Death & Disease

Article Title: 4EBP1 senses extracellular glucose deprivation and initiates cell death signaling in lung cancer

doi: 10.1038/s41419-022-05466-5

Figure Lengend Snippet: A Western blot analysis of 4EBP1 expression in parental A549 and A549-sh4EBP1 cells. B Parental A549 and A549-sh4EBP1 cells were treated with normal or glucose starvation for 48 h. Cell growth rate was assessed by MTT assay. The data represent the averages of three independent experiments (mean ± SD). * P < 0.05. C Parental A549 and A549-sh4EBP1 cells were treated with normal or glucose starvation for 16 h. The changes of cell morphological was investigated (scale bar = 100 µm, magnification: ×100). D Parental A549 and A549-sh4EBP1 cells were treated with normal or glucose starvation for 20 h. Cell apoptosis analysis was done by flow cytometry. E , F In vivo xenograft assay was performed using parental A549 and A549-sh4EBP1 cells, tumors were photographed ( E ), and their weights and volumes were measured ( F ). The P value was calculated by paired t -test. * P < 0.05. G Immunohistochemical staining in the tumor-central region of tumor induced by parental A549 and A549-sh4EBP1 cells for 4EBP1, p-STAT3, BCL2, MCL1, Survivin and Cleaved PARP1 (C-PARP1). Scale bars: 50 μm, magnification: ×200.

Article Snippet: Flow cytometric analysis of apoptotic cells was performed using the Annexin V-FITC / PI Apoptosis Detection Kit (Elabscience).

Techniques: Western Blot, Expressing, MTT Assay, Flow Cytometry, In Vivo, Xenograft Assay, Immunohistochemical staining, Staining

Journal: Oncology Letters

Article Title: Quercetin induces tongue squamous cell carcinoma cell apoptosis via the JNK activation-regulated ERK/GSK-3α/β-mediated mitochondria-dependent apoptotic signaling pathway

doi: 10.3892/ol.2022.13198

Figure Lengend Snippet: Effects of quercetin on cell morphology and viability in human tongue squamous cell carcinoma SAS cells. (A) Cells were treated with quercetin (30 and 50 µM) for 24 h. The morphological changes of cells were observed under an inverted phase-contrast microscope (magnification, ×400). (B) Cells were treated with quercetin (10–300 µM) for 24 h. Cell viability was determined using the MTT assay. (C-a) Apoptosis was determined by staining with the Annexin V-FITC apoptosis assay kit and analyzed using flow cytometry, and quantitative analysis of the (C-b) percentage of phosphatidylserine exposure on the outer cellular membrane and (C-c) early and late apoptotic cells (Annexin V + /PI − and Annexin V + /PI + ) was detected. (D) Cells were treated with quercetin (10–50 µM) for 24 h. Caspase-3 activity was examined by a Caspase-3 Activity Assay Kit. Cisplatin (10 µg/ml) was used as a positive control. In B-D, data are presented as the mean ± standard deviation of four independent experiments with triplicate determination. *P<0.05 vs. vehicle control.

Article Snippet: To assess quercetin-induced apoptosis and necrosis, flow cytometry was performed using the Annexin V-FITC-PI assay kit (BioVision, Inc.).

Techniques: Microscopy, MTT Assay, Staining, Apoptosis Assay, Flow Cytometry, Membrane, Activity Assay, Caspase-3 Activity Assay, Positive Control, Standard Deviation, Control

Journal: Theranostics

Article Title: Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression

doi: 10.7150/thno.27310

Figure Lengend Snippet: Dysregulation of HOXC10 alters the ability of gliomas to induce angiogenesis in vivo . (A) Relative changes in the luminescence of xenograft tumors formed by vector control, HOXC10 -transduced, shRNA control, and HOXC10 -silenced glioma cells. n = 6/group. Error bars, SD. * P < 0.05. (B) Representative bioluminescence images of the indicated tumor-bearing mice. (C) Representative images of H&E staining of glioma sections from the indicated mice. (D) Kaplan-Meier survival curves of mice (n = 6/group) inoculated with the indicated cells. * P < 0.05. (E) CD31 immunohistochemistry staining (arrow) indicating that overexpressing HOXC10 induced, whereas silencing HOXC10 inhibited, glioma angiogenesis in vivo . (F) Quantification of CD31+ microvessel density. Error bars, SD. * P < 0.05. (G) Representative images of the CAM blood vessels stimulated by CM derived from the vector control, HOXC10 -transduced, shRNA control, and HOXC10 -silenced glioma cells. The assay was repeated at least three times with similar results. (H) GSEA plots demonstrating a significant correlation between HOXC10 mRNA expression levels in gliomas and gene signatures of angiogenesis from published datasets (GSE7696). ES, enrichment score; NES, normalized enrichment score.

Article Snippet: IHC was performed using anti-HOXC10 polyclonal antibody (Abcam, ab153904), anti-CD31 polyclonal antibody (Abcam, ab28364), and anti-VEGFA monoclonal antibody (Abcam, ab1316, [VG-1]).

Techniques: In Vivo, Plasmid Preparation, shRNA, Staining, Immunohistochemistry, Derivative Assay, Expressing

Journal: Theranostics

Article Title: Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression

doi: 10.7150/thno.27310

Figure Lengend Snippet: Silencing VEGFA expression or treatment with bevacizumab dramatically inhibits the growth and angiogenesis of tumors overexpressing HOXC10 . (A) Relative changes in the luminescence of xenograft tumors formed by control-shRNA-transduced U251MG/ HOXC10 cells and VEGFA -shRNA-transduced U251MG/ HOXC10 cells. n = 6/group. Error bars, SD, * P < 0.05. (B) Upper panel: representative bioluminescence images of mice bearing tumors formed by control-shRNA-transduced U251MG/ HOXC10 cells and VEGFA-shRNA-transduced U251MG/ HOXC10 cells. Lower panel: representative images of H&E staining of glioma sections from mice bearing the indicated tumors. (C) Kaplan-Meier survival curves of mice (n = 6/group) inoculated with the indicated cells. * P < 0.05. (D) Left panel: CD31 immunohistochemical staining (arrow) indicating that silencing VEGFA inhibited angiogenesis in tumors overexpressing HOXC10 . Right panel: quantification of CD31+ microvessel density. Error bars, SD. * P < 0.05. (E) Relative changes in the luminescence of xenograft tumors formed by HOXC10 -overexpressing U251MG cells treated with IgG or bevacizumab. n = 6/group. Error bars, SD. (F) Upper panel: representative images of mice bearing tumors formed by HOXC10 - overexpressing U251MG cells treated with IgG or bevacizumab. Lower panel: representative images of H&E staining of glioma sections from mice bearing tumors formed by HOXC10 - overexpressing U251MG cells treated with IgG or bevacizumab. (G) Kaplan-Meier survival curves of mice (n = 6/group) bearing tumors formed by HOXC10 -overexpressing U251MG cells treated with IgG or bevacizumab. * P < 0.05. (H) Left panel: CD31 immunohistochemical staining (arrow) indicating that bevacizumab inhibited angiogenesis in tumors overexpressing HOXC10 . Right panel: quantification of CD31 + microvessel density. Error bars, SD. * P < 0.05.

Article Snippet: IHC was performed using anti-HOXC10 polyclonal antibody (Abcam, ab153904), anti-CD31 polyclonal antibody (Abcam, ab28364), and anti-VEGFA monoclonal antibody (Abcam, ab1316, [VG-1]).

Techniques: Expressing, shRNA, Staining, Immunohistochemical staining

Journal: Theranostics

Article Title: Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression

doi: 10.7150/thno.27310

Figure Lengend Snippet: HOXC10 regulates VEGFA promoter activity in glioma cells. (A) The VEGFA promoter luciferase reporter plasmids and Renilla pRL-TK plasmids were transfected into the empty vector-transduced, HOXC10 -transduced, control shRNA-transduced, and the HOXC10 -shRNA transduced glioma cells. Forty-eight hours later, the luciferase signal was examined. (B) Schematic illustration of the cloned fragments of the human VEGFA promoter. (C-D) Luciferase activity of VEGFA promoter fragments in control vector cells and HOXC10 -overexpressing glioma cells. (E-F) Luciferase activity of VEGFA promoter fragments in shRNA control vector cells and HOXC10 -silenced glioma cells. (G) ChIP assays were performed using an anti-HOXC10 antibody to screen three predicted HOXC10-binding sites in the VEGFA promoter. IgG was used as a negative control. (H) ChIP assay showing enrichment of Pol II on region 3 in the VEGFA promoter in the indicated cells. All the assays showed in the figure were independently replicated three times. Error bars, SD. * P < 0.05.

Article Snippet: IHC was performed using anti-HOXC10 polyclonal antibody (Abcam, ab153904), anti-CD31 polyclonal antibody (Abcam, ab28364), and anti-VEGFA monoclonal antibody (Abcam, ab1316, [VG-1]).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, shRNA, Clone Assay, Binding Assay, Negative Control

Journal: Theranostics

Article Title: Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression

doi: 10.7150/thno.27310

Figure Lengend Snippet: HOXC10 interacts with PRMT5 to promote VEGFA transcription. (A) ChIP-qPCR enrichments of histone post-translational modifications on VEGFA promoter region 3 in the indicated cells, displayed using a heatmap. The color key represents an intensity scale for ratio of HOXC10 versus vector or HOXC10 -shRNA versus shRNA control vector, normalized by log2 transformation. (B) ChIP-qPCR enrichments of PRMT5 or lgG on VEGFA promoter region 3 in the indicated cells. (C) ChIP-qPCR enrichments of HOXC10 or lgG on VEGFA promoter region 3 in the control-shRNA-transduced, HOXC10 -overexpressing glioma cells or PRMT5 -shRNA-transduced, HOXC10 -overexpressing glioma cells. (D) Vector or Flag-tagged HOXC10 was transfected into the U251MG or LN229. Co-IP assay showed that HOXC10 interacted with PRMT5. (E) Co-IP assay showed that endogenous HOXC10 interacted with PRMT5 in U251MG and LN229 cells. (F) Far western blotting analysis showed that Flag-tagged HOXC10 in U251MG transfected with Flag-tagged HOXC10 interacted with recombinant GST-PRMT5. (G) Schematic illustration of full length HOXC10 or HOXC10 truncations. (H) Flag-tagged HOXC10 (full length or truncations) and HA-tagged PRMT5 were transfected into U251MG. Co-IP assay revealed that PRMT5 interacted with FL, F1 and F4, but not with F2 and F3. (I) VEGFA promoter luciferase reporter plasmids, Renilla pRL-TK plasmids, vector, or Flag-tagged HOXC10 (full length or truncations) were transfected into U251MG cells. After 48 h, cells were subjected to a luciferase reporter assay. All the assays shown in the figure were independently replicated three times. Error bars, SD. * P < 0.05.

Article Snippet: IHC was performed using anti-HOXC10 polyclonal antibody (Abcam, ab153904), anti-CD31 polyclonal antibody (Abcam, ab28364), and anti-VEGFA monoclonal antibody (Abcam, ab1316, [VG-1]).

Techniques: Plasmid Preparation, shRNA, Transformation Assay, Transfection, Co-Immunoprecipitation Assay, Far Western Blot, Recombinant, Luciferase, Reporter Assay

Journal: Theranostics

Article Title: Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression

doi: 10.7150/thno.27310

Figure Lengend Snippet: PRMT5 and WDR5 are required for HOXC10-mediated VEGFA transcription. (A) ChIP-qPCR enrichments of H3R2me1 and H3R2me2s on VEGFA promoter region 3 in the control-shRNA transduced, HOXC10 -overexpressing glioma cells or PRMT5-shRNA transduced, HOXC10 -overexpressing glioma cells. (B) Histone H3 served as a control for ChIP experiments. (C) ChIP-qPCR enrichments of H3K4me3, polymerase II or IgG on VEGFA promoter region 3 in the vector control, HOXC10 -transduced, control-shRNA transduced, and HOXC10 -shRNA transduced glioma cells. (D) ChIP-qPCR enrichments of WDR5 or IgG on VEGFA promoter region 3 in the vector control, HOXC10 -transduced, control-shRNA transduced, and HOXC10 -shRNA transduced glioma cells. (E) ChIP-qPCR enrichments of H3K4me3, polymerase II or IgG on VEGFA promoter region 3 in the control-shRNA transduced, HOXC10 -overexpressing glioma cells or PRMT5 -shRNA transduced, HOXC10 -overexpressing glioma cells. (F) Real-time PCR analysis showed the VEGFA mRNA expression in the HOXC10 -overexpressing glioma cells treated with vehicle or 1 μM OICR-9429 for 2 days. (G) ChIP-qPCR enrichments of H3K4me3, polymerase II or IgG on VEGFA promoter region 3 in the HOXC10 -overexpressing glioma cells treated with vehicle or 1 μM OICR-9429 for 2 days. All the assays shown in the figure were independently replicated three times. Error bars, SD. * P < 0.05.

Article Snippet: IHC was performed using anti-HOXC10 polyclonal antibody (Abcam, ab153904), anti-CD31 polyclonal antibody (Abcam, ab28364), and anti-VEGFA monoclonal antibody (Abcam, ab1316, [VG-1]).

Techniques: shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing

Journal: Theranostics

Article Title: Overexpression of HOXC10 promotes angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA expression

doi: 10.7150/thno.27310

Figure Lengend Snippet: Clinical relevance of HOXC10 and VEGFA expression in gliomas. (A) HOXC10 levels were positively associated with VEGFA expression in 94 primary human glioma specimens. Left panel: two representative cases are shown. Magnification ×400: scale bars, 50 μm (top 2 rows). Magnification ×1,000: scale bars, 25 μm (bottom 2 rows). Right panel: percentage of glioma specimens showing low or high HOXC10 expression relative to the level of VEGFA. (B) Expression analysis (left) and correlation (right) between HOXC10 expression and VEGFA expression in 10 freshly collected human glioma samples. The western blotting assay was replicated independently three times.

Article Snippet: IHC was performed using anti-HOXC10 polyclonal antibody (Abcam, ab153904), anti-CD31 polyclonal antibody (Abcam, ab28364), and anti-VEGFA monoclonal antibody (Abcam, ab1316, [VG-1]).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Characterization of the novel tumor-suppressor gene CCDC67 in papillary thyroid carcinoma

doi: 10.18632/oncotarget.6709

Figure Lengend Snippet: ( A ) Compared with the control cells (NC), TPC-1 cells with introduced expression of CCDC67 (TPC-1/ CCDC67 ) displayed a significant increase in the G1 phase and decrease in the S phase in the cell cycle. The upper panel shows the distribution of cell cycle phases and the lower panel shows the quantitative measurements of the cell cycle phases. ( B ) Introduced expression of CCDC67 promoted cell apoptosis. The upper panel shows the extent of the apoptotic component of cells and the lower panels showed the quantitative measurements of the apoptotic cells. In the bar graphs, the columns represent mean of at least 3 independent experiments and the little vertical bars on the top of the columns represent SD. ** P < 0.01 in comparison with controls per Student's t -test.

Article Snippet: The dyeing of cells was performed according to the instructions of Annexin V-FITC/PI cell apoptosis detection kit (SouthernBiotech, USA).

Techniques: Control, Expressing, Comparison

Journal: Aging

Article Title: Adipose-derived mesenchymal stem cells-derived exosome-mediated microRNA-342-5p protects endothelial cells against atherosclerosis.

doi: 10.18632/aging.102857

Figure Lengend Snippet: Figure 2. The establishment of the lesion model for HUVECs. (A) The inhibition rate of HUVECs exposed to H2O2 at varied concentrations (0, 500, 1000, 1500 and 2000 uM) were detected by CCK8 assay. (B) HUVECs exposed to H2O2 at varied concentrations (0, 1000, 1500 and 2000 uM) were visualized according to Hoechst staining kit assay. The red arrows indicate nuclear condensation or apoptosis. (C) The apoptosis rate of HUVECs exposed to H2O2 at varied concentrations (0, 1000, 1500 and 2000 uM) were detected by flow cytometry assay. (D) With the enhanced concentrations of H2O2 (0, 1000, 1500 and 2000 uM), the expression levels of cleaved-PARP/PARP, cleaved- caspase3/caspase3, cytochrome C, Bcl-2, Bax and p53 were detected by western blot assay. (E) The corresponding grey-scale maps of (D) were shown. *, P<0.05, **, P<0.01, ***, P<0.001. HUVECs, human umbilical vein endothelial cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: To evaluate the effect of concentrations of H2O2 on cell apoptosis of HUVECs, flow cytometry assay were performed using Annexin V-FITC/PI apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions.

Techniques: Inhibition, CCK-8 Assay, Staining, Flow Cytometry, Expressing, Western Blot

Journal: Aging

Article Title: Adipose-derived mesenchymal stem cells-derived exosome-mediated microRNA-342-5p protects endothelial cells against atherosclerosis.

doi: 10.18632/aging.102857

Figure Lengend Snippet: Figure 3. The screened miR-342-5p facilitated the apoptosis of HUVECs impaired by H2O2. (A) The heat map of top-30 upregulated miRNAs ranked by log2FC value in RNA-sequencing analysis was generated by the online tool ClustVis. (B) The expression levels of ten candidate miRNAs were determined in HUVECs impaired by H2O2 (1500 uM) by qRT-PCR assay. The relative expression of has-miR-342-5p achieved the highest among these ten miRNAs. (C) The expression of miR-342-5p was detected with the gradually increasing concentrations of H2O2 (1000, 1500 or 2000 uM) to treat HUVECs. Results indicated that miR-342-5p expression increased along with the increasing concentration of H2O2. (D) HUVECs were separately transfected with miR-342-5p mimic, inhibitor and their corresponding NC, followed by treating with 1500 uM of H2O2. Their apoptotic rates were then analyzed by flow cytometry assay. The apoptosis rate was increased in miR- 342-5p mimic group (miR-342-5p mimic+H2O2) and decreased in miR-342-5p inhibitor group (miR-342-5p inhibitor+H2O2) when separately compared to their corresponding NC group. (E) HUVECs were separately transfected with miR-342-5p mimic, inhibitor and their corresponding NC, followed by treating with 1500 uM of H2O2. The well-known apoptin (PARP, caspase 3, cytochrome C, Bcl-2, Bax and p53) in treated HUVECs was evaluated by western blot assay. The expression levels of cleaved-PARP/PARP, cleaved-caspase3/caspase3, cytochrome C, and p53 were elevated while the ratio of Bcl-2 to Bax was declined in miR-342-5p mimic group (miR-342-5p mimic+H2O2). Opposite results were obtained in miR-342-5p inhibitor group (miR-342-5p inhibitor+H2O2). (F) The corresponding grey-scale maps of (E) were shown. *, P<0.05, **, P<0.01, ***, P<0.001. MiR-342-5p, microRNA-342-5p; HUVECs, human umbilical vein endothelial cells; FC, fold change; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: To evaluate the effect of concentrations of H2O2 on cell apoptosis of HUVECs, flow cytometry assay were performed using Annexin V-FITC/PI apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions.

Techniques: RNA Sequencing, Generated, Expressing, Quantitative RT-PCR, Concentration Assay, Transfection, Flow Cytometry, Western Blot, Real-time Polymerase Chain Reaction, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Combined inhibition of HER2 and VEGFR synergistically improves therapeutic efficacy via PI3K-AKT pathway in advanced ovarian cancer

doi: 10.1186/s13046-024-02981-5

Figure Lengend Snippet: Synergistic inhibition of OC Cell Proliferation in vitro by RC48 and CM. A A2780, OVCAR-3, SK-OV-3 and HO-8910PM cells were treated with various concentrations of RC48, CM, or their combination (COM) for 72 h. Cell viability was detected using Cell Titer-Glo cytotoxicity assays. B , C The proliferation of A2780, OVCAR-3 cells treated with vehicle control (VEH), 4 μg/mL RC48, 2.5 μM CM, or COM for 60 h was observed using the Incucyte real-time cell analysis system. Images captured at 100 × magnification, respectively. Scale bars = 400 µm. D , E A2780 and OVCAR-3 cells were treated with 4 μg/mL RC48, 2.5 μM CM, or COM for 24 h, and cell proliferation was determined using the EdU assay. Images were captured using a laser scanning confocal microscope. Images were captured at 100 × magnification ( C ) and 400 × magnification ( D ), with scale bars = 400 µm and scale bars = 20 µm, respectively. F , G The anti-proliferative effects were determined by assessing the area of colonies stained with crystal violet. H , I A2780 and OVCAR-3 cells were treated with 4 μg/mL RC48, 2.5 μM CM, or COM for 48 h. Apoptosis was analyzed by staining the cells with Annexin V-FITC/PI and flow cytometry. J , K Western blotting was performed to detect c-Myc, and MCL-1 levels as indicators of apoptotic cell death. GAPDH was used as loading control. The experiments were conducted in triplicate. Data represent the mean ± SEM of three independent experiments. Statistical significance was assessed using one-way ANOVA with Bonferroni post hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: A2780 and OVCAR-3 cells (1 × 10 6 /well) were cultured in 6-well plates and treated with RC48, CM or their combination for 48 h. Flow cytometry was performed using the Annexin V-propidium iodide (PI) apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) on a BD FACSCantoII instrument (USA) to determine cell apoptosis.

Techniques: Inhibition, In Vitro, Control, Cell Analysis, EdU Assay, Microscopy, Staining, Flow Cytometry, Western Blot

Journal: Molecules (Basel, Switzerland)

Article Title: Cell Cycle Arrest and Apoptosis-Inducing Ability of Benzimidazole Derivatives: Design, Synthesis, Docking, and Biological Evaluation.

doi: 10.3390/molecules27206899

Figure Lengend Snippet: Figure 3. The apoptotic effect in A549, MDA−MB 231, and SKOV3 cancerous cells after treatment with vehicle control and compounds 10 and 13 by annexin V−FITC/PI staining using flow cytometry. One-way ANOVA was used to test for statistical differences (* p < 0.05, ** p < 0.01).

Article Snippet: Apoptosis Analysis The assessment of apoptosis was performed using the Annexin V-FITC/PI analysis Kit, Cell Signaling Technology (CST), as instructed by the manufacturer [37].

Techniques: Control, Staining, Cytometry